MjYT of Methanocaldococcus jannaschii

The last of the four components analyzed during this study was the corresponding tRNA of the aaRS MjYRS (Ch. 4.1.3) and its derivatives, called MjYT. MjYT, and the corresponding aaRS, were available in two different versions, where one was decoding the amber stop codon UAG (MjYT_CUA; pCLA5) and the other the frameshift codon AGGA (MjYT_UCCU;

pCLA6).

Figure 4.13: Predicted secondary structure of MjYT_CUA.

The structure was obtained from ViennaRNA Web Services^[156]. The region to which the probes C40 and C41 bind, that is the anticodon stem and in parts the D-loop, is highlighted in red.

For the detection of these tRNAs on northern blots we developed hybridization probes covering the same regions of the tRNA as shown for PylT (Figure 4.7), namely the anticodon stem and in parts the D-loop. The oligos C40 and C41 (Table 8.2) were designed to anneal to the stop codon tRNA as shown in Figure 4.13.

Next, we extracted RNA from E. coli cells (Ch. 3.2.3.14) containing plasmids for either MjYRS_TAG only (pCLA2) or in combination with the cognate tRNA (pCLA2 and pCLA5).

Additionally, RNA was isolated from cells transformed with a plasmid that held both components at once but in which the aaRS was decoding for p-benzoyl-L-phenylalanine^[83]

(BPA; pCLA21) instead of tyrosine. Total RNA was separated with acid urea PAGE and its size was estimated by size standards (Ch. 3.1.4) and T7RP transcribed M. jannaschii Tyr-tRNA (T7 TyrT) as depicted in Figure 4.14-A.

Figure 4.14: Detection of MjYT_CUA in total RNA extracts.

A) Acid urea PAGE for the size estimation of isolated RNA from cell cultures harboring plasmids for MjYRS_TAG (pCLA2; 2.8 µg loaded onto gel), MjYRS_TAG/MjYT_CUA (pCLA2 and pCLA5; 2.7 µg loaded) and BPARS/MjYT_CUA (pCLA21; 3.0 µg loaded). The latter one was grown in LB medium supplemented with 1 mM BPA. T7 TyrT (T7T) with a size of 77 nt served as a positive control (0.8 µg loaded). Two different size standards consisting of DNA and RNA nucleotides were used to consider different running behavior. ONL = Oligonucleotide Ladder; RL = Low Range ssRNA Ladder. The gel was soaked in a “gel red” bath (1:10,000 in H2O (v/v)) and separated DNA/RNA bands were visualized by UV light using a gel documentation machine. B) Northern blot of samples described in A). A total amount of 1.4 µg RNA from cells with MjYRS_TAG only was loaded. In contrast 1.35µg RNA from cells with MjYRS_TAG and MjYT_CUA and 1.5 µg RNA from cells with BPARS/MjYT_CUA (+1 mM BPA) were loaded. The DIG labeled probe C41 was utilized for hybridization and chemiluminescence signals recorded as described in Ch. 3.2.3.14.

We observed a signal between 70 and 80 nt in the lane for the T7 TyrT (Figure 4.14-A). The actual size of the tRNA is 77 nt. Total RNA extracts from cells yielded the expected smear.

The northern blot of the same samples (Figure 4.14-B) showed two prominent bands. The upper one was present in all lanes and the lower one only if the cells were transformed

with the plasmid for the tRNA. Since the lower one only appeared in samples with the tRNA and the size correlated to the signal seen in Figure 4.14-A, we assigned this band as MjYT_CUA.

The appearance of the second upper band was thought to be an unspecific binding of the probe to other RNA fragments, especially other tRNAs, due to its presence in all lanes. Thus, we blasted the sequence of the probe against the genome of E. coli (using the program BLASTN 2.2.29+^[157], data not shown). It became apparent that the last 13 bases of the probe used for the northern blot were complementary to several kinds of E. coli asparagine tRNAs. Hence, new probes binding specifically to the M. jannaschii tRNA had to be developed and tested. Figure 4.15 shows an alignment of the two versions of MjYT, CUA and UCCU, together with those asparagine tRNAs to which the probe C41 was hybridizing.

In addition to the tRNAs, all different probes tested for a specific binding to MjYT were aligned, those were MjYT_AGGA_DIG_Probe1-4 (C44 to C47). We switched to the frameshift codon version of MjYT since this tRNA was used in further experiments in combination with the UAG decoding tRNA PylT.

Figure 4.15: Sequence alignment of MjYT.

The DNA sequences of both MjYT versions (CUA = pCLA5 and UCCU = pCLA6) were compared to northern blot hybridization probes MjYT_AGGA_DIG_Probe1-4 (C44 to C47) and E. coli tRNAs Asn, Asn1, Asn2, Asn3a and Asn3b.

Subsequent northern blots revealed that MjYT_AGGA_DIG_Probe3 (C46; Figure 4.16-A) was the only hybridization probe that was able to bind to the tRNA MjYT_UCCU specifically without giving background signals from E. coli Asn-tRNAs (Figure 4.16-B). The other probes yielded very weak signals, no signals at all, or exhibited unspecific binding (not shown).

Furthermore, Probe3 could discriminate between the two versions of MjYT, only giving signals if the frameshift variant was present in the cells. Comparable to PylT (Figure 4.9-C) we observed two bands at different heights. Since this only happened if the corresponding aaRS was present, the upper signal was assigned as the aminoacylated form of MjYT_UCCU, again.

Figure 4.16: Refinement of MjYT detection.

A) Predicted secondary structure of MjYT_UCCU. The structure was obtained from ViennaRNA Web Services^[156]. The region to which the probe MjYT_AGGA_DIG_Probe3 (C46) binds is highlighted in red. B) Northern blot from the refinement studies using probes MjYT_AGGA_DIG_Probe1-4 (C44 to C47). In this case only MjYT_AGGA_DIG_Probe3 (C46) is shown. Approximately 0.090 µg RNA were loaded for each sample. After hybridization, chemiluminescence signals were recorded as described in Ch. 3.2.3.14. The upper band (MjYT^Tyr) represents the aminoacylated form of MjYT_UCCU, whereas the lower band (MjYT) depicts the uncharged form.